Lymphocytes can be activated by antigens, leading to immune responses, or inactivated or eliminated, leading to tolerance toward the antigen. Tolerance to self-antigens is a fundamental property of the immune system. Failure to establish tolerance by the immune system leads to autoimmune diseases.
New therapies for the treatment of autoimmune diseases include generating or giving a specific type of lymphocytes, regulatory T cells (Tregs), to patients. Tregs suppress the activation and effector functions of other, self-reactive and potentially pathogenic lymphocytes, which results in suppressed immune responses and maintenance of self-tolerance. Tregs are a subset of CD4+ T cells. The best characterized Tregs are CD4+ CD25+FoxP3+ T cells. Tregs can be generated by self antigen recognition in the thymus and by antigen recognition in peripheral lymphoid organs. Dendritic cells (DCs) are the most potent antigen presenting cells. DCs participate in the innate immunity and the acquired immune response. DCs perform many functions for the immune system such as: 1) uptake, processing, and presentation of antigens, 2) activation of effector cells such as T-cells, B-cells and NK-cells, and 3) secretion of cytokines and other immune-modulating molecules to direct the immune response. DCs recognize specific pathogens and various danger signals. Recognition of pathogen-derived products and danger signals mediated by specific receptors on DCs initiates the process of maturation, which can be further modified by inflammatory stimuli or T cell-derived signals. Maturation is a process when activated DCs undergo morphological, phenotypic, and functional changes that culminate in complete transition from antigen-capturing cells to fully mature antigen presenting cells (APC). Maturation is characterized by increased expression of costimulatory molecules such as CD40, CD80, and CD86, MHC-upregulation, the loss of the capacity to take up and process antigens and the production of wide spectrum of inflammatory cytokines and chemokines (IL-1β, IL-6, IL-8, and IL-12). Once activated, DCs migrate to the lymph nodes where they interact with T cells and B cells to initiate and shape the adaptive immune response.
DCs derive from the myeloid lineage of hematopoietic cells. Myeloid progenitors in bone marrow give rise to macrophage-DC progenitors, which further differentiate into common/myeloid DCs and plasmacytoid DCs. Monocytes can also differentiate into DCs. Differentiation gives rise to immature dendritic cells (iDCs). The primary function of iDCs is to capture and process antigens. Immature DCs can mature as they contact and process antigens in an inflammatory environment. A variety of factors can induce maturation following antigen uptake and processing within DCs, including: whole bacteria or bacterial-derived antigens (e.g. lipopolysaccharide, LPS), inflammatory cytokines, ligation of select cell surface receptors (e.g. CD40) and viral products (e.g. double-stranded RNA). Bacterial-derived antigens and viral products can be recognized via Toll-like receptor (TLR). TLRs recognize various components of invading pathogens. Ligand binding to the TLRs on DCs induces proinflammatory cytokine production and enhanced antigen presentation to naive T cells, and thus activates antigen-specific adaptive immune responses. Distinct TLR ligands provide distinct activation status and cytokine production patterns for antigen presenting cells, resulting in the induction of differential immune responses. Thus, TLRs are critical molecules to fine-tuned adaptive immune responses depending on invading pathogens. Given that stimulation by TLR ligands can fine-tune the immune response toward specific pathogens, it is important for therapeutic vaccination that the DC properties are maintained once the cells are administrated to the patient.
DCs can also be partially maturated resulting in upregulation of MHC and costimulatory molecules and lymph node homing capacity, but lacking proinflammatory cytokine production. Such DCs have been termed semi-mature DCs (Lutz et al. 2002. Trends Immunol 23:445-449).
Tolerogenic DCs (tolDCs) are antigen presenting cells with immunosuppressive properties. They can induce tolerance through the presentation of antigen with inadequate co-stimulation and cytokine production for effector cell activation. TolDCs are commonly defined by low or intermediate levels of MHCII, costimulatory molecules CD80, CD86 and CD40, and chemokine receptor CCR7, in addition to a remarkably increased antigen uptake capacity. TolDCs express high levels of inhibitory molecules such as Ig-like transcripts (ILT) molecules (ILT3/ILT4) and/or PD-L-molecules (PD-L1, PD-L2). Additionally, tolDCs secrete low amounts of proinflammatory cytokines (IL-12p70) and high quantities of anti-inflammatory cytokines, such as IL-10. TolDCs induce T cell anergy, T cell suppression and the generation of regulatory T cells by several mechanisms, including conversion of naïve T cells into Tregs, release of immunosuppressive cytokines, and expression of functional indoleamine-2,3 dioxygenase (IDO). TolDCs are generally considered as semi-mature DCs. Several signaling pathways involved in the induction and maintenance of immunosuppressive role of tolDCs have been identified. The pro-inflammatory DC maturation is normally associated with the activation of numerous signaling pathways including transcription factors NF-κB and p38 MAPK (Nakahara et al. 2006, J Derm Science 42: 1-11; Katholnig et al. 2013, J Immunol 190: 1519-1527). The pattern of activated signaling events triggered in tDCs is profoundly different and involves the activation of ERK1/2, non-canonical NF-κB pathway, STAT3 and IDO (Qian et al. 2006, Blood 108: 2307-2315; Harden et al. 2012, Immunol Invest 41: 738-764; Manches et al. 2012, PNAS 109: 14122-14127; Farias et al. 2013, CNS 19: 269-277).
Expression of the indoleamine 2,3-dioxygenase in tolDCs and the ensuing production of tryptophan metabolites has been shown to induce direct suppression of effector T-cell activity and concurrent expansion of Tregs (Harden et al. 2013. Immunol Invest 41:738-764). TolDCs can be generated from precursor cells in vitro and represent potentially promising tool for a specific form of cell-based therapy for induction or restoring immune tolerance in the context of transplantation and autoimmune diseases (Fischbach et al. 2013. Sci Transl Med 5:179ps7). Different approaches that target DC differentiation and function by various mechanisms have been shown to establish a tolDC phenotype (Naranjo-Gomez et al. 2011. J Transl Med 9:89; Li et al. 2007. J Immunol 178:5480-7; Torres-Aguilar et al. 2010. J Immunol 184:1765-75). Notably, Dexamethasone and/or vitamin D3 receptor agonists (VDR; 1,25(OH)2D 3 and its analogues) have been described to generate tolDCs through the suppression of NF-κB-dependent DC maturation (Adorin et al. 2009. Handb Exp Pharmacol 251-73; van Hooten et al. 2009. Handb Exp Pharmacol 233-49). Such Dex/VitD3 conditioned tolDCs have been shown to acquire a robust immunoregulatory phenotype and are currently tested in early clinical trial in patients with rheumatoid arthritis (Stoop et al. 2010. Arthritis Rheum 62:3656-65). TolDCs can also be generated from DCs conditioned with 19-nor-1,25-dihydroxyvitamin D2 (paricalcitol), the analogue of the active form of vitamin D2 (Sochorova et al. 2009. Clin Immunol 133:69-77). Other approaches to establish a tolDCs phenotype have also involved the use of neuropeptides (vasoactive intestinal peptide or pituitary adenylate cyclase-activating polypeptide) (Chorny et al. 2005. Proc Nat Acad Sci 102:13562-7; Chorny et al. 2006. Blood 107:3787-84; Gonzalez-Rey et al. 2006. Gastroenterology 131:1799-811) or the mTOR inhibitor rapamycin (Haidinger et al. 2010. J Immunol 185:3919-31). None of cells produced by these methods have been demonstrated to have the characteristics of the Dex/vitamin D2 tolDCs described herein.
One of the major concerns associated with therapeutic vaccination with in vitro established tolDCs is their functional stability. Once injected into patients, tolDCs must retain highly stable tolerogenic properties in the absence of tolerogenic agents. A potential risk of ex-vivo prepared tolDCs is that their application to an organism with chronic inflammation, such as autoimmune disease, may switch them to an activated phenotype when encountering proinflammatory signals in vivo. This might than contribute to the further expansion of the autoimmune reaction and would be detrimental for the outcome of the treatment.